Iron (II) is an important molecule to pathogens. Iron (II) can be acquired from Iron (III), the more readily available form, by reductase activity. According to Brown et al, 2002, Rev Microbes and Infection 4(11): 1149-1156, Iron (II) can be utilized by most pathogens for the purpose growth and virulence. Previously, genetic studies by Kadner, et al, J. Bacteriol. 143:256-264. (1980), show that drug resistance, in Eschericia coli, to iron containing antibiotics, can result from gene mutations in its iron uptake genes.
The invention relates to a preparation comprising X-S-Cys-X peptide moiety, hereon referred to as BRAUNMYCINÎ>>, (where Î>> is equal to X-S-Cys-X.) for iron-reductase activity; and methods of obtaining purified iron-binding, iron-reductase, moiety, using a iron-nitriloacetic acid jaw, Fe3+ or 2+-NTA, IBPJAW, hereon, referred to as BRAUNMYCIN8567, as an accessory, in batch affinity chromatography; and the interpretation of this activity, like its use in detecting resistance to iron-containing antibiotics in Deoxyribonucleic acid, or DNA, producing cells. X, as used above, maybe either or both of glycan sugars, or peptide sequences >0.12 kDa in length.
The present invention was developed from studies utilizing genetic information on pneumococcal genetic sequences relating to iron acquisition genes previously made public through the NCBI gene Bank database, under gene accession number AF338658.1; GI 18478640 at http://www.tigr.org (2002), by Olisa, N. R., et al, reported earlier In Abstracts of The 100th General Meeting of the American Society for Microbiology B-22, p.45 (2000) by the same author. The present invention, has been developed to function as an accessory in batch affinity chromatography.
The present invention was evaluated using a Streptococcus pneumoniae wild type Rx1 strain, a spontaneous albomycin resistant strain alb−, isolated from Rx1, and a genetically induced albomycin resistant strain, created as a result of the reverse genetics of a major pneumococal iron acquisition gene, pia−in Rx1. The choice of S. pneumoniae as a model, stems from the fact that the organism in question displays a scrupulous need for iron (II). According to studies performed by Tai et al, 1993, Infect. Immun. 61:5401-1087, S. pneumoniae lack siderophore. Based on this information, theoretically, they should have well-developed cell wall iron-reductase for use in iron acquisition.
The present invention was used to purify unique X-S-Cys-X peptides from cell wall iron-reductase in Rx1. The inability of the present invention to purify same sequences in mutant strains, formed the scientific basis for detecting albomycin resistance in alb−, and pia− mutants. Albomycin, is an iron-containing antibiotic, and natural siderophore produced by Streptomyces spp. In example, we use albomycin, as a marker, for iron-reductase activity, since killing by the antibiotic occurs when iron present in its molecules are reduced or taken up.
The uniqueness of the present invention was determined after extensive databases searches in the achieves of the US patent and trademark office, using the key words iron-reductase, and iron-nitriloacetic acid. When iron-reductase was used in search, results yielded a single insignificant match. It appears that Zenno et al, 1995, was awarded a U.S. Pat. No. 5,468,631 for a gene encoding an enzyme, flavin, with reducing activity and nitroreductase activity. This gene, the enzyme, as well as their techniques are unrelated to ours. The pure form of the functional X-S-Cys-X moiety from iron-reductase, targeted by our preparation, may or may not contain glycan sugars. Furthermore, our technique is not genetic, it is a batch affinity protocol, and computational proteomics that uses, a unique JAW to capture glycan sugar containing peptides of the S-Cys, mass spectrometry sequencing, and database searches. Finally, in reference to the database search, there were no matches using the key word iron-nitriloacetic acid.